Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Abstract

Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella, showing disruption of supercoiling near the terminus and replicon failure at 42 degrees C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella. Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo.

Strategies for introducing the gyrB652 C-to-A transversion mutation into the E. coli chromosome. Shown is a map of the gyrB-yidB region of E. coli and the gyrB652 mutation (marked by “A”). (Strategy A) A synthetic, single-stranded, 70-mer oligonucleotide (oligonucleotide 1 [#1]) with the C-to-A transversion at position 36. (Strategy B) A PCR product made with oligonucleotides 1 and 2 to generate a 339-bp fragment with 35 bp of homology upstream and 303 bp downstream of the C/A substitution. (Strategy C) Oligonucleotides 5 and 6 make a 2,824-bp PCR fragment with the gyrB652 mutation at bp 54, with 1,160 bp of further gyrB homology followed by a selectable Kanr module and 133 bp of intergenic homology. (D) Control PCR. Oligonucleotides 5 and 7 make a 2,916-bp PCR product, with 1,306 bp of WT gyrB homology, with “C” marking the WT gyrB sequence, the Kanr module, followed by 133 bp of downstream homology. Arrow tips with genes indicate transcription directions.

Stabilities of plasmid pGem vectors carrying the gyrB652 or WT gyrB gene of S. enterica serovar Typhimurium in E. coli. LB medium containing 50 μg/ml Amp was inoculated 1:100 in triplicate with overnight cultures of E. coli containing a pGem plasmid with the Salmonella gyrB652 gene (NH3706) or a WT Salmonella gyrB gene (NH3707). Cultures were grown at 30°C to stationary phase in a shaking incubator. A portion of culture (labeled 1 to 3) was serially diluted into 96-well microtiter plates, and 3-μl aliquots were spotted onto LB plates with and without 50 μg/ml Amp. The dilution factor is shown on the right side. Pictures were taken after 24 h of incubation at 30°C.

Stability of plasmid pAG111, which expresses a WT E. coli gyrB gene, in Salmonella and E. coli. LB media containing 50 μg/ml Amp was inoculated with overnight cultures at 1:100 and grown at 30°C for 4 h in a shaking incubator with the indicated amounts of IPTG. A portion of each culture was serially diluted into 96-well microtiter plates, and 3 μl aliquots were spotted onto LB plates as indicated. The dilution factor is shown at the right. Pictures were taken after 24 h of incubation at 30°C.

E. coli and Salmonella supercoiling analysis of pBR322 on 2-D chloroquine gels. Salmonella (NH3715) and E. coli (NH3718) with WT topoisomerases were grown to mid-log phase (70 Klett units). Plasmid pBR322 DNA was purified, concentrated, and then separated in a 25-cm 1.0% agarose gel. Electrophoresis was for 46 h at 2.0 V/cm in 2 μM chloroquine for the first dimension and 46 h at 2.0 V/cm containing 20 μM chloroquine for the second dimension (). The supercoil densities determined by the band counting method were −0.059 for Salmonella and −0.069 for E. coli.

E. coli and Salmonella supercoil comparison of a plasmid containing 56 bp of alternating GC repeats (pRW478) on a 2-D chloroquine gel. Strains of Salmonella (NH3713) and E. coli (NH3716) with WT topoisomerases and pRW478 were grown to mid-log phase (70 Klett units). Plasmid DNA was purified, and topoisomers were resolved in a 25-cm 1.0% agarose gel containing 2 μM chloroquine for 46 h at 2.0 V/cm in the first dimension and for 46 h in 20 μM chloroquine for the second dimension. More-negatively supercoiled topoisomers migrate to the upper-right region of this gel system. The pattern for E. coli shows a second distribution of highly supercoiled molecules with a break in the distribution (B/Z transition breaks in the downward sector of the arc in the right panel). The second very highly supercoiled distribution indicates the formation of Z-DNA in vivo ().

Species-specific phenotypes for ΔseqA strains grown in rich medium. M9 medium containing 0.2% glucose, 1 μg/ml thiamine, and 40 μg/ml l-proline was inoculated with single colonies and grown overnight at 30°C in a shaking incubator. OD600 was measured, and cultures were diluted to an OD600 of 1.0. A portion of each culture was serially diluted (1:10) into 96-well microtiter plates, and 3-μl aliquots were spotted onto both LB plates and M9 plates with the above-mentioned additives. Pictures were taken after 24 h of incubation at 30°C.

Species-specific phenotypes for ΔmukB strains grown in rich medium. Minimal medium containing 0.2% glucose was inoculated with single colonies and grown 48 h at room temperature in a shaking incubator. OD600 was measured, and cultures were diluted to an OD600 of 1.0. A portion of each culture was serially diluted (1:10) into 96-well microtiter plates, and 3-μl aliquots were spotted onto both LB and minimal medium-glucose plates. Pictures were taken after 48 h of incubation at 30°C.

Different Mu lytic profiles for E. coli and Salmonella. Mu lysogens of E. coli (NH1126) (open symbols) and Salmonella (NH742) (closed symbols) were diluted 1:100 in duplicate LB cultures and grown in a 30°C incubator to log phase (OD600 = 0.4). The cultures were shifted to a 42°C shaking water bath, and the OD600s and numbers of CFU were measured at 10-min intervals. The E. coli cultures (open squares) began lysis at 30 min, and the Salmonella cultures (filled squares) began lysis at 40 min. The number of viable cells in Salmonella cultures (filled circles) dropped 50-fold, while for E. coli (open circles), viability dropped by 8 orders of magnitude.